The overall goal of this project is to develop and validate a novel class of fluorescent sensors for paramagnetic metal ions (PMIs, e.g., Fe2+, Fe3+, Mn2+ and Mn3+), and to use these sensors to provide deeper insight into the uptake and homeostasis of PMIs in bacteria and the role of PMIs in pathogenesis. PMIs are essential elements for both humans and bacteria; the availability of these metal ions is sharply limited for pathogens, as a part of a host defense mechanism known as ?nutritional immunity?; the most well characterized examples being Fe and Mn sequestration during infection. Moreover, Fe and Mn-regulated pathways are closely linked with pathways involved in managing oxidative stress, as occurs in phagocytic respiratory burst. Despite the importance of PMIs in nutritional immunity and oxidative stress pathways, the precise mechanisms dictating nutritional immunity, bacterial uptake of PMIs, and the ability of certain bacterial strains to circumvent metal starvation and thrive are unclear. A major barrier to understanding these complex mechanisms is the lack of spatiotemporal detection of PMIs in their different OSs in living bacterial cells. This proposal seeks to overcome this major barrier by selection and characterization of PMI-specific DNAzymes, and subsequent development and validation of DNAzyme-based turn-on fluorescent sensors selective not only for different PMIs, but also different oxidation states of the same PMI in two model systems (Staphylococcus aureus and Escherichia coli). Specifically, we plan to employ in vitro selection to obtain DNAzymes with high cleavage activity and strong affinity for different PMIs (Fe2+ and Mn2+), while maintaining specificity for the different oxidation states of the same metal ion (Fe2+ vs. Fe3+, and Mn2+ vs. Mn3+). Biochemical studies of these DNAzymes will provide information about conserved sequences, pH and metal ion dependence, and kinetic parameters of the DNAzyme activity. Biophysical characterization using spectroscopic methods (UV-vis and EPR) and x-ray crystallography will elucidate PMI-binding stoichiometry, affinity and selectivity in these DNAzymes. The knowledge acquired will be used to convert these DNAzymes into PMI sensors using the patented catalytic beacon technology. The use of a ?caged? and FRET DNAzyme sensor enabling quantitative monitoring of metal ion concentration and speciation in living cells under temporal control will also be explored. Since pathogenic bacteria such as S. aureus and E. coli are a major public health issue, especially due to the spread of antibiotic resistance, our ability to develop turn-on fluorescent sensors for the real time detection of PMIs in cells will overcome a major barrier within the field of nutritional immunity by improving our understanding of the uptake and homeostasis of PMIs in bacteria and the role of PMIs in pathogenesis. Ultimately, knowledge gained from these sensors could provide insights necessary to develop novel strategies to fight against bacterial infection.